Natriuretic peptide receptor-A (NPR-A), also known as guanylyl cyclase-A, is a transmembrane receptor guanylyl cyclase that is activated by the cardiac hormones atrial natriuretic peptide and B-type natriuretic peptide. Although ligand-dependent NPR-A degradation (also known as downregulation) is widely acknowledged in human and animal models of volume overload, downregulation in cultured cells is controversial. Here, we examined the effect of ANP exposure on cellular NPR-A levels as a function of time. Relative receptor concentrations were estimated using guanylyl cyclase and immunoblot assays in a wide variety of cell lines that endogenously or exogenously expressed low or high numbers of receptors. ANP exposures of 1 h markedly reduced hormone-dependent but not detergent-dependent guanylyl cyclase activities in membranes from exposed cells. However, 1-h ANP exposures did not significantly reduce NPR-A concentrations in any cell line. In contrast, exposures of greater than 1 h reduced receptor concentrations in a time-dependent manner. The time required for half of the receptors to be degraded (t1/2) in primary bovine aortic endothelial and immortalized HeLa cells was approximately 8 h. In contrast, a 24-h exposure of ANP to 293T cells stably overexpressing NPR-A caused less than half of the receptors to be degraded. To our knowledge, this is the first report to directly measure NPR-A down-regulation in endogenously expressing cells. We conclude that down-regulation is a universal property of NPR-A but is relatively slow and varies with receptor expression levels and cell type.